Cassette Transporter ABCB1 Research Articles

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6.

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible “bulge” loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

Molecular and Pharmacological Inhibition of ATP Binding Cassette Transporter ABCB6 Decreases Chemoresistance in Acute Myeloid Leukemia

Cytarabine (Ara-C) and Daunorubicin (Dnr) remain the main stay treatment for patients with acute myeloid leukemia (AML). Drug resistance and relapse are the major hurdles in achieving long term event-free survival. ATP Binding Cassette transporter proteins (ABC) plays a prominent role in drug resistance by extruding the anticancer drugs from the cells. Among the mitochondrial ABC transporters, the heme protoporphyrin transporter ABCB6 was found to be highly expressed in cell lines- HT-29, A549, SK3, St-4, A2780, SKOV3, KK47, T24, K562, and U937 made resistant to camptothecin, cisplatin, etoposide (VP-16), adriamycin and also in HepG2 and Hep3B cell lines resistant to arsenite. We have previously reported that ABCB6 expression was significantly higher in primary AML samples resistant to Dnr (Varatharajan et al., 2017). AML patients with FLT3 -ITD showed low ABCB6 expression levels and were more sensitive to arsenic trioxide (ATO) compared to FLT3 wild type (Abraham et al., 2014). In the present study, using a molecular and inhibitor based approach we explored the functional role of ABCB6 in mediating resistance to Dnr and ATO in AML.Cell lines K562 and THP1 with high ABCB6 expression and high IC50 to both Dnr and ATO were transduced with an inducible lentiviral shRNA targeting ABCB6 . The efficiency of knock down was confirmed by real time PCR, immunoblot and immunofluorescence. As ABCB6 is known to localize in the mitochondrial membrane, its role in mediating reactive oxygen species (ROS) levels were assessed by flowcytometry using DCFDA. The effect of ABCB6 knock down in causing apoptosis to Dnr and ATO mediated apoptosis was evaluated using 7AAD and Annexin V. The effect of ABCB6 pharmacological inhibitor, tomatidine in improving chemosensitivity was also evaluated in combination with Dnr and ATO by MTT assay. The efficiency of knock down was >80% in K562 (p<0.001) and >60% in THP1 (p<0.001) cell lines. Concordant to the mRNA levels, there was a significant decrease in ABCB6 protein levels by western blot and immunofluorescence. Interestingly, ABCB6 knock down increased basal ROS levels in both K562 (18 fold) and THP1 (3.1 fold) compared to their respective doxycycline negative (Dox -ve) control. The combination of ABCB6 knockdown and Dnr treatment increased ROS by 2.1-fold in THP1 and by 21-fold in K562 cells and along with ATO by 1.7-fold in THP1 and 5.7-fold in K562 compared to the Dox -ve cells. Knockdown of ABCB6 also improved sensitivity to 0.125µM of Dnr (17.45% to 36.94%, p= ns) and 6µM ATO (10% to 39%, p=0.007). ABCB6 inhibitor tomatidine reduced the IC50 values of THP1 to Dnr (0.16µM to 0.05µM) and ATO (>6µM to 2.91µM) and K562 cells to ATO (12.08µM to 7.26µM). Our results suggest that targeting ABCB6 could sensitize different subgroups of AML to treatment with ROS inducing agents like Dnr and ATO. Further mechanistic studies are required to investigate if the effect of ABCB6 inhibition is selective to leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Isolation and sex steroid effects on the expression of the ATP-binding cassette transporter ABCB6 in Harderian glands of hamster (Mesocricetus auratus)

ATP-Binding Cassette, subfamily B, member 6 (ABCB6) is a transporter that is upregulated by elevated intracellular porphyrin concentrations. In the Harderian gland (HG), the synthesis of porphyrins appears to be under the influence of gonadal steroids and to exhibit a dimorphic pattern. To explore whether ABCB6 is also influenced by sex steroids, we isolated its specific cDNA sequence and investigated its mRNA levels in the HGs of hamsters. ABCB6's cDNA sequence presents an open reading frame (ORF) of 2529 bp that encodes a predicted 842-amino acid (aa) protein with a molecular weight of 93 kDa. Multiple sequence alignments showed that ABCB6's aa sequence is highly conserved and shares the highest homology (93%) with mouse ABCB6. RT-qPCR analysis indicated that ABCB6 is expressed in all the tissues examined, exhibiting high expression levels in the liver, adrenal glands, and testis. The mRNA concentrations of ABCB6 in HGs were very similar between males and in females; similarly, gonadectomy and treatment with sex steroids appear to scarcely affect ABCB6 mRNA levels. The intraglandular content of ABCB6 mRNA showed discrete, though non-significant, variations through the estrous cycle. The results provide evidence that gonadal steroids have a minimal physiological role on the regulation of ABCB6 expression and might indicate that this transporter has a small effect on porphyrin trafficking in the HGs of hamsters.

Erythrocytes lacking the Langereis blood group protein ABCB6 are resistant to the malaria parasite Plasmodium falciparum

The ATP-binding cassette transporter ABCB6 was recently discovered to encode the Langereis (Lan) blood group antigen. Lan null individuals are asymptomatic, and the function of ABCB6 in mature erythrocytes is not understood. Here, we assessed ABCB6 as a host factor for Plasmodium falciparum malaria parasites during erythrocyte invasion. We show that Lan null erythrocytes are highly resistant to invasion by P. falciparum, in a strain-transcendent manner. Although both Lan null and Jr(a-) erythrocytes harbor excess porphyrin, only Lan null erythrocytes exhibit a P. falciparum invasion defect. Further, the zoonotic parasite P. knowlesi invades Lan null and control cells with similar efficiency, suggesting that ABCB6 may mediate P. falciparum invasion through species-specific molecular interactions. Using tandem mass tag-based proteomics, we find that the only consistent difference in membrane proteins between Lan null and control cells is absence of ABCB6. Our results demonstrate that a newly identified naturally occurring blood group variant is associated with resistance to Plasmodium falciparum.

Roles of aldo-keto reductases 1B10 and 1C3 and ATP-binding cassette transporter in docetaxel tolerance

Docetaxel (DTX) is widely used for treatment of inveterate lung and prostate cancers, but its continuous administration elicits the hyposensitivity. Here, we established the DTX-resistant variants of human lung cancer A549 and androgen-independent prostate cancer Du145 cells and found that the resistance development provoked aberrant up-regulations of aldo-keto reductase (AKR) 1B10 and AKR1C3 in A549 and Du145 cells, respectively. In addition, the sensitivity to the DTX toxicity was significantly decreased and increased by overexpression and knockdown of the two AKR isoforms, respectively. Furthermore, the resistant cells exhibited a decreased level of reactive 4-hydroxy-2-nonenal formed during DTX treatment, and the decrease was alleviated by adding the AKR inhibitors, inferring that the two AKRs confer the chemoresistance through elevating the antioxidant properties. The development of DTX resistance was also associated with enhanced expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with inhibitors of the two AKRs and ABCB1 additively sensitized the resistant cells to DTX. Intriguingly, the AKR1B10 inhibitor also suppressed the lung cancer cross-resistance against cisplatin. The results suggest that combined treatment with AKRs (1B10 and 1C3) and ABCB1 inhibitors exerts overcoming effect against the cancer resistance to DTX and cisplatin, and can be used as the adjuvant therapy.

Abstract 2529: Expression of ABCF1 with stem cell markers CD133/GEP/beta-catenin in fetal liver and liver cancer cells

Abstract Understanding stem cell biology in relation to cancer is of growing importance with the cancer stem cells (CSCs) notion and recurring deregulated stem cell pathways reported in cancers. ATP-binding cassette (ABC) transporter ABCB1 (also named P-glycoprotein and MDR1) has shown to be CSC marker, mediate multidrug-resistance in model systems though with controversial clinical relevance. We have systemically examined the expression profiles of ABC genes in hepatocellular carcinoma (HCC) clinical samples. ABCF1 was significantly over-expressed in HCCs compared to the adjacent non-tumor liver tissues (P&amp;lt;0.001) and associated with recurrence-free survival after curative partial hepatectomy (log rank, P = 0.010). Recurrences have conventionally been attributed to aggressive cancer features and, recently, CSC features, as CSCs in small load could grow to tumor bulk. We therefore further explored the significance of ABCF1 in both fetal liver and liver cancer cells in respect to stem cell signaling. Expression of ABCF1 in fetal, neonatal and adult mouse livers were examined by immunohistochemistry, western blot and flow cytometry. Consistently, elevated ABCF1 expression was demonstrated in early embryonic stages [embryonic day 13 (E13): 42.4%; E15: 24.6%] but rarely in neonates and adult livers [1.5% and 1.2%, respectively]. Also, ABCF1 co-stained with stem cell-related marker β-catenin and hepatic CSC markers CD133 and GEP in fetal hepatocytes and in HCC cells (Table 1). ABCF1 suppression by siRNA enhanced chemo-sensitivity of the HCC cells Hep3B compared to control cells on cisplatin (cell apoptosis 7.5% vs 1.7%, P&amp;lt;0.001) and 5-Fluorouracil (11.7% vs 4.7%, P&amp;lt;0.001). Subcellular fractionation analysis on HCC cells showed that ABCF1 protein was present in both the cytoplasmic and membrane fractions. The current study highlighted the functional significance of ABCF1 in liver development and liver cancer as stem cell-related molecule regulating chemo-resistance. Table 1.Co-expression of ABCF1 and hepatic stem cell markers in mouse fetal livers and HCC cellsFetal liver cells (E15)Liver cancer cells (Hep3B)ABCF1+24.6%34.5%CD133+16.3%88.8%ABCF1+ CD133+20.9%34.3%GEP+45.7%65.7%ABCF1+ GEP+24.0%29.7%β-catenin+40.6%20.9%ABCF1+ β-catenin+13.5%17.5% Citation Format: Sze Wai Fung, Phyllis Fung Yi Cheung, Chi Wai Yip, George Sai Wah Tsao, Siu Tim Cheung. Expression of ABCF1 with stem cell markers CD133/GEP/beta-catenin in fetal liver and liver cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2529.

Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport.

Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2μM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport.

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.

ET-72 * THE STRATEGY STUDY OF ENHANCED ALA-INDUCED PpIX FLUORESCENT QUALITY IN MALIGNANT GLIOMAS

OBJECTIVE: Gliomas are characterized by its local invasiveness and poor prognosis. It has been demonstrated that the survival benefit is associated with the extent of surgical resection. But it is difficult to distinguish tumor margins due to the infiltrative nature, and gliomas often recur within 2-3 cm from the borders of the resection cavity. Recently, fluorescence-guided resection has been demonstrated to improve discrimination of tumor tissue and increase the extent of surgical resection. However, insufficient protoporphyrin IX accumulation may limit the benefits of FGR in low-grade glioma and the marginal area. METHODS: We investigated the expression of the ATP-binding cassette transporter ABCB6 in gliomas, and the correlation between ABCB6 expression and PpIX accumulation. Meanwhile, Glioma cells and astrocytes were preconditioned with calcitriol or arsenic trioxide. Changes in ALA-induced PpIX fluorescence were assessed by flow cytometry and fluorescence microscopy. Furthermore, expression of porphyrin synthetic enzymes in pretreated glioma cells was analyzed. RESULTS: The expression levels of ABCB6 significantly elevated in gliomas, A previously finding was the fact that ABCB6 mRNA expression in solidly fluorescing tumor tissues was significantly higher than that in vaguely fluorescing tumors, Accordingly, ABCB6 overexpression in cell lines increased ALA-induced PpIX accumulation and fluorescent quality. Meanwhile, calcitriol or arsenic trioxide can be administered prior to ALA as a non-toxic preconditioning regimen to significantly enhance ALA- induced PpIX levels and fluorescence. This increase in PpIX level was detected preferentially in glioma versus normal cells. Moreover, mechanistic studies documented that expression of the porphyrin synthesis enzymes coproporphyrinogen oxidase was increased by calcitriol or arsenic trioxide at the mRNA level. CONCLUSIONS: ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimising the subjective discrimination of vague fluorescence. We demonstrated for the first time a simple, non-toxic and highly effective preconditioning regimen to selectively enhance PpIX fluorescence in glioma cells.

Abstract 766: Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364

Abstract The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. Plk1 inhibitors BI 2536, volasertib and GSK461364, were designed to selectively inhibit cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to Plk1 inhibitors is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that in human cancer cells, the overexpression of ABCB1 and/or ABCG2 can lead to acquired resistance to three selective Plk1 inhibitors, BI 2536, volasertib and GSK461364. Moreover, these Plk1 inhibitors stimulate the ATPase activity of ABCB1 and ABCG2, as well as competitively inhibit the drug substrate transport mediated by ABCB1 and ABCG2. More significantly, the reduced chemosensitivity and Plk1 inhibitors-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor of ABCB1 and ABCG2. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to Plk1 inhibitors, a combined regimen of Plk1 inhibitors and modulators or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic. Citation Format: Chung-Pu Wu, Hong-May Sim, Suresh V. Ambudkar. Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2014-766

Abstract LB-222: Chemical and structural flexibility contribute to the broad substrate and modulator specificity of multidrug resistance-linked P-glycoprotein (ABCB1).

Abstract The human ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein, P-gp) mediates efflux of cytotoxic anticancer agents from cells, thereby conferring resistance to chemotherapeutic agents in most cancers. One of the most important features of ABC drug transporters, including P-gp, is their ability to recognize a variety of chemically dissimilar hydrophobic or amphipathic compounds. In this report, we elucidate the molecular basis of the promiscuous nature of this transporter. We modeled the structure of human P-gp using the X-ray structure of mouse P-gp as a template, with docked substrates (cyclosporine A, valinomycin and nilotinib) and modulators (tariquidar and 5′-Fluorosulfonylbenzonyl 5′-adenosine) in the pocket where the QZ59-peptide inhibitors bind mouse P-gp. Single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, M949, F978, V982 and A985 residues at the drug binding site with cys in a cys-less P-gp were expressed in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cys-less wild-type P-gp. Several drugs including cyclosporine A, valinomycin, nilotinib and tariquidar partially or completely lose the ability to inhibit the photolabeling of mutant P-gps with the transport substrate [125I]-Iodoarylazidoprazosin, indicating that these drugs cannot bind at their primary binding site. However, the drugs can modulate the ATP hydrolysis of the mutant P-gps, demonstrating that they bind at alternate sites. The transport of six fluorescent substrates in HeLa cells overexpressing triple mutants (Y307C/Q725C/V982 and Y307A/Q725A/Y953A) is also not significantly altered, showing that substrates bound at alternate sites are still transported. In aggregate, our results suggest that a large flexible pocket in the transmembrane domains in P-gp is able to bind chemically diverse compounds through a versatile allocation of hydrogen-bond donors and acceptors and hydrophobic residues. When residues of the primary drug-binding site are mutated, substrates and modulators bind to alternate sites on the transporter, revealing an exceptional chemical flexibility. We propose that both the chemical and structural flexibility provide the molecular basis for interaction of P-gp and possibly other ABC drug transporters with a wide range of substrates and modulators. Citation Format: Suresh V. Ambudkar, Eduardo E. Chufan, Khyati Kapoor, Stewart R. Durell, Hong-May Sim. Chemical and structural flexibility contribute to the broad substrate and modulator specificity of multidrug resistance-linked P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2013-LB-222

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Increased Expression of ABCB6 Enhances Protoporphyrin IX Accumulation and Photodynamic Effect in Human Glioma

Glioma recurrence usually occurs close to the tumor resection margins as a result of residual infiltrating glioma cells. 5-aminolevulinic acid (ALA) fluorescence-guided resection of gliomas has been demonstrated to enhance discrimination of tumor tissue and to improve survival. ALA-based photodynamic therapy is an effective albeit still experimental adjuvant treatment option for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the benefits of fluorescence-guided resection and photodynamic therapy. We investigated the expression of the ATP-binding cassette transporter ABCB6, which regulates porphyrin synthesis, in surgical specimens from human gliomas and manipulated ABCB6 in human glioma cell lines. Our findings demonstrated that expression levels of ABCB6 were greatly elevated in human gliomas compared with normal brain tissues and correlated with World Health Organization histologic grade. A previously undescribed finding was that ABCB6 mRNA expression in solidly fluorescing tumor tissues was higher than that in vaguely fluorescing tumors, suggesting that ABCB6 may be at least in part responsible for PpIX accumulation in glioma cells. Accordingly, ABCB6 overexpression in glioma cell lines caused a marked increase in intracellular levels of PpIX, and was more sensitive to ALA-induced photodynamic therapy-events that could be prevented by silencing ABCB6 via siRNA treatment. Our findings indicate a crucial role of ABCB6 in ALA metabolism and accumulation of PpIX in glioma. ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimizing the subjective discrimination of vague fluorescence and improving the efficacy of ALA-based photodynamic therapy.

Abstract 689: Short-term exposure of tumor cells to chemotherapeutic agents is associated with changes in microRNA expression

Abstract The acquisition of resistance to anticancer drugs is a key obstacle to successful cancer therapy. An increasing number of studies have investigated the roles of microRNAs (miRs) in drug resistance. We reported that the upregulation of miR-135b/-196b correlated positively with acquired drug resistance as well as expression of the ATP-binding cassette (ABC) transporter ABCB1 in the human T-cell leukemic CCRF-CEM cell line (T-T Ho et al, Proceedings of the 101st Annual Meeting of the AACR 2010). We now report that when we exposed cells to etoposide for 48 h, we found similar increases in miR-135b/-196b in leukemias (RPMI 8226 [myeloma], HL-60 [acute promyelocytic leukemia], Jurkat and MOLT-4 [both acute T cell leukemia]), but not in solid tumors (MCF7 breast cancer and A2780 ovarian cancer), suggesting that induction of expression of these miRs may be histiotype-specific. To develop a mechanistic understanding of differentially expressed miRs, we have studied miR-196b, which maps between the HOXA9 and HOXA10 genes on chromosome 7p15.2. We observed upregulation of HOXA9 mRNA in relation to short-term chemotherapeutic drug exposure in CCRF-CEM cells, indicating that the expression of miR-196b is linked to HOXA gene transcription. To investigate the kinetics of expression of miR-135b/-196b, ABCB1 and ABCC1, we have performed rechallenge experiments in which CCRF-CEM cells were repeatedly exposed to etoposide for 48 h with a 3-day drug-free incubation in between. We found that changes in miR-135b/-196b expression were paralleled by expression of the ABCB1 gene, but not the ABCC1 transporter gene. In addition, subsequent etoposide (IC50, 300 nM) rechallenges (2nd and 3rd challenge, days 5 and 10) led to progressively attenuated expression of the miR-135b/-196b and ABCB1 compared to that seen after the first challenge. These results further support the observation that miR-135b/-196b expression is associated with ABCB1 upregulation, and suggest that miR-135b/-196b are involved not only in the initial response of cancer cells to chemotherapeutic challenge, but also in the development of chemoresistance via mechanisms associated with expression of efflux pump genes. We consequently measured expressions of miR-135b/-196b, ABCB1 and ABCC1 in CCRF-CEM cells exposed to repeated drug challenge after either 15 or 20 passages in drug-free medium. Treatment of these previously-exposed cells again with 300 nM etoposide resulted in unexpected attenuation of miR-135b/-196b and ABCB1 expression. We further determined that the etoposide IC50 in these cells had increased to 1 µM. Our results offer insights into the establishment of the drug-resistant phenotype and suggest that induction of miR-135b/-196b and the drug-resistant phenotype is transiently acquired, but its loss after drug exposure seems to re-set the IC50 to higher levels. (Supported in part by 5RO1CA40570 from NCI to WTB, and in part by UIC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 689. doi:10.1158/1538-7445.AM2011-689

The ATP-Binding Cassette Transporter ABCB6 Is Induced by Arsenic and Protects against Arsenic Cytotoxicity

Arsenic, an environmental carcinogen, remains a major public health problem. Arsenic damages biological systems through multiple mechanisms, including the generation of reactive oxygen species. ABCB6 is an ATP-binding cassette transporter that is highly expressed in cells resistant to arsenic. We have recently demonstrated that ABCB6 expression protects against cellular stressors. In the present study, we evaluated the significance of ABCB6 expression to arsenic toxicity both in mice and in cell culture. We show that sodium arsenite induces ABCB6 expression in a dose-dependent manner both in mice fed sodium arsenite in drinking water and in cells exposed to sodium arsenite in vitro. Arsenite-induced ABCB6 expression was transcriptionally regulated, but this induction was not mediated by the redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrate that, in HepG2 and Hep3B cells, knockdown of ABCB6 expression using ABCB6-specific small interfering RNA sensitized the cells to arsenite toxicity. In contrast, stable overexpression of ABCB6 conferred a strong survival advantage toward arsenite-induced oxidative stress. Collectively, these results, obtained by both loss of function and gain of function analysis, suggest that ABCB6 expression in response to sodium arsenite might be an endogenous protective mechanism activated to protect cells against arsenite-induced oxidative stress.

Human ABCB6 Localizes to Both the Outer Mitochondrial Membrane and the Plasma Membrane

Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.

Characterization of Heme as Activator of Toll-like Receptor 4

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.

The ATP-binding cassette transporter ABCG2 (BCRP) and ABCB1 (MDR1) variants are not associated with disease susceptibility and disease phenotype in Hungarian patients with inflammatory bowel diseases

The ATP-binding cassette transporter ABCG2 (BCRP) and ABCB1 (MDR1) variants are not associated with disease susceptibility and disease phenotype in Hungarian patients with inflammatory bowel diseases

ATP-binding cassette transporter ABCG2 (BCRP) and ABCB1 (MDR1) variants are not associated with disease susceptibility, disease phenotype response to medical therapy or need for surgeryin Hungarian patients with inflammatory bowel diseases

Objective. MDR1 (ABCB1), a member of the ATP-binding cassette (ABC) transporters, is an attractive candidate gene for the pathogenesis of inflammatory bowel diseases (IBD) and perhaps for response to therapy. Since limited data are available on MDR1 and ABCG2 polymorphisms in East European IBD patients, the aim of this study was to investigate ABCG2 and MDR1 variants and responses to medical therapy and/or disease phenotype in Hungarian patients. Material and methods. A total of 414 unrelated IBD patients (Crohn's disease (CD): 265, age: 35.2±12.1 years, duration: 8.7±7.6 years and ulcerative colitis (UC): 149, age: 44.4±15.4 years, duration: 10.7±8.9 years) and 149 healthy subjects were investigated. ABCG2 G34A, C421A and MDR1 C3435T, G2677T/A single nucleotide polymorphisms (SNPs) were detected using real-time polymerase chain reaction (PCR). Detailed clinical phenotypes were determined by reviewing the medical charts. Results. The frequency of the ABCG2 and MDR1 SNPs was not significantly different among IBD, CD, UC patients and controls. There was no difference in risk for steroid resistance in CD patients carrying variant ABCG2 (19.6% versus non-carriers 18.4%, p=NS) or MDR1 3435T (CC: 22.2% versus CT/TT: 17.6%) alleles. In addition, carriage of the variant allele was not associated with disease phenotype, presence of extra-intestinal manifestations, smoking, response to infliximab therapy or the need for surgery. In UC, the carriage of variant ABCG2 alleles seemed to be preventive for arthritis (15.5% versus 31.7%, OR: 0.39, 95% CI: 0.16–0.98). Conclusions. MDR1 and ABCG2 SNPs were not associated with disease susceptibility or disease phenotype in Hungarian patients, and variant alleles did not predict the response to medical therapy or the need for surgery. Further studies are needed to clarify the association between the presence of ABCG2 variants and arthritis in UC.

Identification of a mammalian mitochondrial porphyrin transporter

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.